Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications

Author(s): Emmert-Buck MR, Ahram M, Flaig M, Gillespie JW, Duray P, Linehan WM, Ornstein D, Niu S, Zhao Y, Petricoin EF

Publication: Proteomics, 2003, Vol. 3, Page 413

PubMed ID: 12687609 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to compare results of 2D electrophoresis of protein extracted from case-matched specimens that were differentially preserved by snap freezing in OCT, or fixation in ethanol or formalin followed by paraffin embedding.

   Summary of Findings:

   Protein extracted from formalin-fixed, paraffin-embedded (FFPE) specimens produced few protein spots in comparison to the hundreds produced with OCT snap frozen specimens. While the protein distribution of ethanol-fixed, paraffin-embedded (EFPE) specimens was similar to that observed with OCT snap frozen specimens and protein identification by mass spectrometry was successful, EFPE tissue sections generated reduced protein yield (by 25%), fewer protein spots (500 versus 700 spots per tissue section, respectively), and an increased incidence of artifacts signifying poor protein quality compared to OCT-embedded snap frozen controls. While increasing the number of EFPE tissue sections used for extraction increased the intensity of protein spots accordingly it did not increase the number of observable spots, nor was the number or quality of protein spots affected by variations to the extraction protocol.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Ethanol
   • OCT
   • Formalin

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels
   Protein	GC-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Ethanol OCT Formalin (buffered)
   Biospecimen Aliquots and Components	Aliquot size/volume	One 5 um section Multiple 5 um sections
   Analyte Extraction and Purification	Analyte isolation method	70 degrees C for 2 h Boiling No heat treatment Protein extraction buffer with 0.25% SDS Protein extraction buffer without SDS Cold acetone No acetone

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2. Study Purpose

   The purpose of this study was to compare protein yields and banding patterns of case-matched stained and microdissected cells that were either OCT-embedded and snap frozen or fixed in ethanol and embedded in paraffin.

   Summary of Findings:

   Similar protein patterns were observed following microdissection of ethanol-fixed and fresh frozen specimens, although total protein yield and the number of corresponding proteins spots were approximately 50% lower among patient-matched ethanol-fixed specimens compared to those that were frozen. The number of protein spots among differentially preserved specimens was comparable when the amount of protein loaded into the gel was standardized by increasing the number of microdissected ethanol-fixed cells used for protein extraction in relation to the number of frozen cells. Differential protein expression of normal and tumor regions within EFPE specimens appear to be preserved, as the protein profiles of adjacent regions containing normal epithelial cells or invasive prostate carcinoma cells were 97% similar, with differential expression observed in only 3% of the proteins evaluated; of these, thioredoxin peroxidase was upregulated and tropomyosin beta was down regulated in tumor cells. Tropomyosin beta protein expression determined by 2D gel electrophoresis was confirmed by immunohistochemistry, in which expression was limited to normal epithelial and smooth muscle cells and not infiltrating tumor cells.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Ethanol
   • OCT

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Ethanol OCT
   Immunohistochemistry Specific	Targeted peptide/protein	Tropomyosin beta

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3. Study Purpose

   The purpose of this study was to determine to assess the analytical impact of using prestained tissue sections for protein extraction and separation. Tissue specimens that were ethanol-fixed, paraffin-embedded and stained with hematoxylin, eosin, or both were compared to unstained controls. Protein yield and banding pattern were also compared in ethanol-fixed, paraffin-embedded specimens that were deparaffinized in xylene or a xylene substitute (Sub-X).

   Summary of Findings:

   Compared to protein extracted from unstained EFPE tissue sections, protein extracted from sections that were stained with eosin alone or in conjunction with hematoxylin were shifted to the acidic end of the 2D gel. Proteins extracted from sections stained only with hematoxylin displayed an identical distribution to those from unstained sections. Although data was not shown, the authors report identical results when specimens were deparaffinized in xylene or the xylene substitute, Sub-X.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Ethanol

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   1D/2D gels Specific	Type of tissue stain	Unstained Hematoxylin Eosin Hematoxylin and eosin
   Analyte Extraction and Purification	Deparaffinization	Xylene Xylene substitute (Sub-X)

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